EP1268784B1 - An entzündlichen darmerkrankungen beteiligte gene und deren verwendung - Google Patents

An entzündlichen darmerkrankungen beteiligte gene und deren verwendung Download PDF

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EP1268784B1
EP1268784B1 EP01919586A EP01919586A EP1268784B1 EP 1268784 B1 EP1268784 B1 EP 1268784B1 EP 01919586 A EP01919586 A EP 01919586A EP 01919586 A EP01919586 A EP 01919586A EP 1268784 B1 EP1268784 B1 EP 1268784B1
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seq
nucleic acid
sequence
polypeptide
dna
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EP1268784A2 (de
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Jean-Pierre Hugot
Gilles Thomas
Mohamed Zouali
Suzanne Lesage
Mathias Chamaillard
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Fondation Jean Dausset-CEPH
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to genes involved in inflammatory and / or immune diseases and some cancers, particularly inflammatory cryptogenic diseases of the intestine, as well as protein encoded by these genes. Diagnostic methods of inflammatory diseases are also objects of the present invention.
  • IBD Cryptogenic Inflammatory Bowel Diseases
  • UC ulcerative colitis
  • TM Crohn's disease
  • IBD is a chronic disease that evolves throughout life and which affect about 1 to 2 people per 1000 inhabitants in Western countries, which represents between 60,000 and 100,000 patients in France. These are diseases appearing in the young subject (the peak of incidence is in the third decade), evolving by thrusts interrupted by remissions, with complications such as undernutrition, stunting in children, bone demineralization and ultimately malignant degeneration to breast cancer colon. There is no specific treatment. The usual therapies make anti-inflammatory drugs, immunosuppressive drugs and surgery. All these Therapeutic means are themselves a source of iatrogenic morbidity important. For all these reasons, IBD appears as an important public health problem.
  • IBD Intradetiology
  • Factors are involved in the occurrence of the disease as in witness the secular increase in disease incidence and concordance incomplete in monozygotic twins.
  • the only risk factors environmental conditions currently recognized are 1) tobacco whose role is harmful in MC and beneficial in UC and 2) appendectomy that has a role protector for the RCH.
  • IBD African and family aggregations of these diseases.
  • IBD are more common in the Caucasian population and especially the population Jewish Central European. Family forms account for 6 to 20% of cases of IBD. They are particularly common when the onset of the disease is early.
  • twins it was studies in twins that allowed confirm the genetic character of these diseases. Indeed, the concordance rate between twins for these diseases is more important in monozygotic twins than in dizygotic twins pleading strongly for a hereditary component to IBD, especially MC.
  • IBDs are complex genetic diseases involving several different genes, interaction with each other and with environmental factors. IBD can therefore classified in the context of multifactorial diseases.
  • the present invention relates to the highlighting of the sequence nucleic acid genes involved in IBD, and other inflammatory diseases, as well as the use of these nucleic sequences.
  • chromosome 16 for several immune and inflammatory diseases such as spondyloarthritis ankylosing, Blau's syndrome, psoriasis, etc. (Becker et al., 1998; al., 1996). All these diseases could then share a single gene (or a same group of genes) located on chromosome 16.
  • the maximum genetic linkage test is almost always located at the same position, at D16S409 or D16S411 separated only by 2cM. This result is in contrast with the large size (usually greater than 20cM) of the confidence interval attributable to the genetic location according to a approach using nonparametric linkage analysis.
  • CM genes known in the pericentromeric region of the chromosome 16, such as the interleukin-4 receptor, CD19, CD43, CD11, appear as good potential candidates for CM. Preliminary results do not plead however not in favor of the implication of these genes in the CM.
  • the present invention provides the sequence not only of IBD1 gene, but also the partial sequence of another gene, called IBD1prox because of its location near IBD, and highlighted as reported in the examples below.
  • IBD1prox the partial sequence of another gene
  • the peptide sequence expressed by the IBD1 and IBD1prox genes is represented by SEQ ID No. 2 and SEQ ID No. 5 respectively; the sequence genomic of these genes is represented by SEQ ID NO: 3 and SEQ ID NO: 6 respectively.
  • nucleic acid nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, sequence nucleotide, terms that will be used interchangeably in the present description means a precise sequence of nucleotides, modified or no, to define a fragment or a region of a nucleic acid, with or without unnatural nucleotides, and which may correspond as well to a double-stranded DNA, a single-stranded DNA that transcripts said DNAs.
  • the nucleic sequences according to the invention encompass also PNA (Peptid Nucleic Acid), or the like.
  • sequences nucleotides in their natural chromosomal environment that is, in the state natural. These are sequences that have been isolated and / or purified, that is, they are were taken directly or indirectly, for example by copying, their environment has been at least partially modified. We also hear designate the nucleic acids obtained by chemical synthesis.
  • variant nucleic sequence is also intended to mean any RNA or CDNA resulting from a mutation and / or variation of a splice site of the sequence genomic nucleic whose cDNA has the sequence SEQ ID No. 1.
  • the invention preferably relates to purified or isolated nucleic acid according to the present invention, characterized in that it comprises or consists of the sequence SEQ ID No. 1, of its complementary sequences or sequences of the RNA corresponding to SEQ ID No. 1.
  • Primers or probes characterized in that they comprise a sequence of a nucleic acid according to the invention, are also part of the invention.
  • the present invention also relates to primers or probes according to the invention which can in particular make it possible to highlight or discriminate variant nucleic sequences, or identify the sequence genomic genes whose cDNA is represented by SEQ ID NO: 1 using in particular an amplification method such as the PCR method, or a related method.
  • the invention also relates to the use of an acid sequence nucleic acid according to the invention as probe or primer, for the detection, identifying, assaying or amplifying nucleic acid sequence.
  • the polynucleotides that can be used as a probe or as a primer in methods of detection, identification, dosing or nucleic acid amplification have a minimum size of 15 bases, preferably 20 bases, or more preferably 25 to 30 bases.
  • Probes and primers according to the invention can be labeled directly or indirectly by a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable signal and / or quantifiable.
  • the unlabeled polynucleotide sequences according to the invention can be used directly as a probe or primer.
  • Sequences are usually marked to obtain sequences usable for many applications. Marking of primers or probes according to the invention is carried out by radioactive elements or by non-radioactive molecules.
  • the non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenine, haptens, dyes, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents.
  • the polynucleotides according to the invention can thus be used as primer and / or probe in processes using in particular the technique of PCR (polymerase chain amplification) (Rolfs et al., 1991).
  • This technique requires the choice of oligonucleotide primer pairs flanking the fragment which needs to be amplified.
  • Amplified fragments can be identified, for example after agarose gel electrophoresis or polyacrylamide, or after a chromatographic technique such as filtration on gel or ion exchange chromatography, then sequenced.
  • the specificity of the amplification can be controlled using as primers the sequences nucleotides of polynucleotides of the invention and as templates, plasmids containing these sequences or the derived amplification products.
  • the Amplified nucleotide fragments can be used as reagents in hybridization reactions to highlight the presence, in a sample of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.
  • the invention also relates to the nucleic acids that can be obtained by amplification using primers according to the invention.
  • amplification techniques of the target nucleic acid can be advantageously used as an alternative to PCR (PCR-like) with the help of pair of nucleotide sequence primers according to the invention.
  • PCR-like one intended to designate all methods using direct reproductions or nucleic acid sequences, or in which the systems marking have been amplified, these techniques are of course known. In general it is the amplification of the DNA by a polymerase; when the original sample is an RNA it is first necessary to perform a reverse transcription.
  • the target polynucleotide to be detected is an mRNA
  • the cDNA obtained will then serve as a target for the primers or the probes implemented in the amplification or detection method according to the invention.
  • the hybridization technique of probes can be carried out in various ways (Matthews et al., 1988).
  • the most general method is to immobilize the acid nucleic acid extracts cells from different tissues or cells in culture on a (such as nitrocellulose, nylon, polystyrene) and to incubate, in well-defined conditions, target nucleic acid immobilized with the probe. After hybridization, the excess of probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity related to the probe).
  • nucleic probes according to the invention can be used as capture probes.
  • a probe called a “capture probe”
  • a probe is immobilized on a support and serves to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected by means of a second probe, called a “detection probe”, marked by an element easily detectable.
  • antisense oligonucleotides that is to say whose structure ensures, for example, hybridization with the target sequence, inhibition of product expression corresponding.
  • sense oligonucleotides which, by interaction with proteins involved in the regulation of product expression corresponding, will induce either an inhibition or an activation of this expression.
  • the oligonucleotides of the invention can be used in vitro and in vivo.
  • the present invention also relates to an isolated polypeptide characterized in that it comprises a polypeptide corresponding to SEQ ID NO: 2
  • polypeptide means proteins or peptides.
  • the present invention also relates to the cloning vectors and / or expression comprising a nucleic acid or coding for a polypeptide according to the invention.
  • a vector may also contain the elements necessary for expression and optionally secretion of the polypeptide in a host cell.
  • a host cell is also an object of the invention.
  • Vectors characterized in that they comprise a sequence of promoter and / or regulator according to the invention, are also part of the invention.
  • Said vectors preferably comprise a promoter, signals initiation and termination of translation, as well as appropriate transcription regulation. They must be stable in the cell and may possibly have particular signals specifying the secretion of the translated protein.
  • control signals are chosen according to the host used cell.
  • the nucleic acid sequences according to the invention can be inserted into autonomously replicated vectors within the host chosen, or integrative vectors of the chosen host.
  • the vectors viral infections including adenoviruses (Perricaudet et al., 1992), retroviruses, lentiviruses, poxviruses or herpesviruses (Epstein et al. 1992).
  • adenoviruses Perricaudet et al., 1992
  • retroviruses lentiviruses
  • poxviruses poxviruses
  • herpesviruses Epstein et al. 1992.
  • the person skilled in the art knows the technologies that can be used for each of these systems.
  • viruses are, for example, retroviruses (Temin, 1986), or AAVs (Carter, 1993).
  • naked polynucleotides such as the naked DNA or the naked RNA according to the technique developed by the company VICAL, the artificial chromosomes of bacteria (BAC, bacterial artificial chromosome), Yeast Artificial Chromosomes (YACA, yeast artificial chromosome) for expression in yeast, artificial chromosomes of mice (MAC, mouse artificial chromosome) for expression in murine cells and so favorite artificial chromosomes of man (HAC, human artificial chromosome) for expression in human cells.
  • BAC bacterial artificial chromosome
  • YACA yeast Artificial Chromosomes
  • MAC mouse artificial chromosome
  • HAC human artificial chromosome
  • Such vectors are prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into a host appropriate by standard methods, such as for example lipofection, electroporation, heat shock, transformation after permeabilization chemical membrane, cell fusion.
  • the invention furthermore comprises the host cells, in particular the cells eukaryotes and prokaryotes transformed by the vectors according to the invention as well as transgenic animals, preferably mammals, except humans, comprising one of said transformed cells according to the invention.
  • transgenic animals preferably mammals, except humans, comprising one of said transformed cells according to the invention.
  • These animals can be used as models, for the study of the etiology of diseases inflammatory and / or immune diseases, and in particular inflammatory diseases of the digestive tract, or for the study of cancers.
  • bacterial cells Olins and Lee, 1993
  • yeast cells Buckholz, 1993
  • animal cells particularly mammalian cells
  • mammalian cells Edwards and Aruffo, 1993
  • cells chinese hamster ovary CHO
  • insect cells in which processes using, for example, baculovirus (Luckow, 1993).
  • a preferred cellular host for the expression of proteins of the invention consists of COS cells.
  • mammals according to the invention animals such as rodents, in particular mice, rats or rabbits, expressing a polypeptide according to the invention.
  • animals are also preferred.
  • animals such as mice, rats or rabbits, characterized in that the gene coding for the protein of sequence SEQ ID No. 2 or whose sequence is coded by the homologous gene in these animals, is not functional, is invalidated or has at least one mutation.
  • transgenic animals are obtained for example by recombination homologue on embryonic stem cells, transfer of these stem cells to embryos, selection of affected chimeras at the lineage level reproducers, and growth of said chimeras.
  • transgenic animals according to the invention can thus overexpress the gene coding for the protein according to the invention, or their homologous gene, or expressing said gene into which a mutation is introduced.
  • These animals transgenics, in particular mice, are obtained for example by transfection copy of this gene under the control of a strong promoter of ubiquitous nature, or selective tissue type, or after viral transcription.
  • the transgenic animals according to the invention can be rendered deficient for the gene encoding one of the SEQ sequence polypeptides ID No. 2 or its homologous genes, by inactivation using the LOXP / CRE recombinase system (Rohlmann et al., 1996) or any other system inactivation of the expression of this gene.
  • the cells and mammals according to the invention can be used in a method of producing a polypeptide according to the invention, as described below, and can also be used as a model of analysis.
  • Cells or mammals transformed as previously described can also be used as models to study interactions between polypeptides according to the invention, and the chemical or protein compounds involved directly or indirectly in the activities of the polypeptides according to the invention, this to study the different mechanisms and interactions involved.
  • polypeptides according to the invention can in particular be used for the selection of products interacting with the polypeptides according to the invention, in particular the protein of sequence SEQ ID No. 2 or its variants according to the invention, cofactor, or inhibitor, especially competitive, or having an activity agonist or antagonist of the activity of the polypeptides according to the invention.
  • said transformed cells or transgenic animals are used for particularly for the selection of products to combat pathologies related to an abnormal expression of this gene.
  • the invention also relates to the use of a cell, a mammal or a polypeptide according to the invention for the screening of chemical compounds or biochemicals that may interact directly or indirectly with the polypeptides according to the invention, and / or capable of modulating the expression or the activity of these polypeptides.
  • the invention also relates to a method for screening compounds capable of interacting in vitro or in vivo with a nucleic acid according to the invention, by using a nucleic acid, a cell or a mammal according to the invention, and by detecting the formation of a complex between the candidate compounds and the nucleic acid according to the invention.
  • the invention also relates to the use of a nucleic acid sequence according to the invention for the synthesis of recombinant polypeptides.
  • the method of producing a polypeptide of the invention in form recombinant, itself included in the present invention is characterized by that the transformed cells, in particular the cells or mammals of the present invention, under conditions allowing the expression of a polypeptide recombinant encoded by a nucleic acid sequence according to the invention, and that recovering said recombinant polypeptide.
  • the recombinant polypeptides characterized in that they are susceptible to be obtained by the said method of production, are also part of the invention.
  • the recombinant polypeptides obtained as indicated above can both in glycosylated and non-glycosylated form and may present or not the natural tertiary structure.
  • sequences of the recombinant polypeptides can also be modified to improve their solubility, especially in aqueous solvents.
  • polypeptides can be produced from the acid sequences nucleic acid defined above, according to the techniques for producing polypeptides recombinants known to those skilled in the art.
  • the acid sequence used nucleic acid is under the control of signals allowing its expression in a cellular host.
  • An efficient system for producing a recombinant polypeptide requires to have a vector and a host cell according to the invention.
  • These cells can be obtained by introducing into host cells of a nucleotide sequence inserted into a vector as defined above, and then culturing said cells under conditions allowing replication and / or the expression of the transfected nucleotide sequence.
  • the methods used for the purification of a recombinant polypeptide are known to those skilled in the art.
  • the recombinant polypeptide can be purified from lysates and cell extracts, the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific monoclonal or polyclonal antibodies, etc.
  • polypeptides according to the present invention can also be obtained by chemical synthesis using one of the many known peptide syntheses, for example techniques using solid phases (see in particular Stewart et al., 1954) or techniques using partial solid phases, by condensation of fragments or by synthesis in conventional solution.
  • Polypeptides obtained by chemical synthesis and which may comprise corresponding non-natural amino acids are also included in the invention.
  • Mono- or polyclonal antibodies or their fragments, antibodies chimeric or immunoconjugate complexes, characterized in that they are capable of specifically recognize a polypeptide according to the invention, are part of the invention.
  • polyclonal antibodies can be obtained from a serum of an animal immunized against the polypeptides according to the invention, in particular produced by genetic recombination or peptide synthesis, depending on the usual procedures.
  • Mono- or polyclonal antibodies or their fragments, antibodies chimeric or immunoconjugate complexes, characterized in that they are capable of specifically recognize the polypeptides of sequence SEQ ID No. 2 are particularly preferred.
  • Specific monoclonal antibodies can be obtained according to the conventional method of hybridoma culture described by Köhler and Milstein (1975).
  • the antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') 2 fragments. They can also be in the form of immunoconjugates or labeled antibodies in order to obtain a detectable and / or quantifiable signal.
  • the invention also relates to methods for detecting and / or purification of a polypeptide according to the invention, characterized in that they an antibody according to the invention.
  • the invention further comprises purified polypeptides, characterized in that that they are obtained by a method according to the invention.
  • the antibodies of the invention in particular monoclonal antibodies, can also be used for the detection of these polypeptides in a sample organic.
  • polypeptides according to the invention in particular the polypeptides of sequence SEQ ID No. 2 or one of its variants, on specific tissue sections, for example by immunofluorescence, gold, enzymatic immunoconjugates.
  • the antibodies of the invention can be advantageously implemented in any situation where the expression of a polypeptide according to the invention, normal or mutated, must be observed.
  • the antibodies according to the invention can also be used in the treatment of an inflammatory and / or immune disease, or cancer, at when an abnormal expression of the IBD1 gene is observed.
  • Abnormal expression means overexpression or expression of a mutated protein.
  • the invention indeed provides the IBD1 gene sequence involved in inflammatory and / or immune diseases, and in particular IBD.
  • One of the teachings of the invention is to specify the mutations in these Nucleic or polypeptide sequences, which are linked to a phenotype corresponding to one of these inflammatory and / or immune diseases.
  • mutations can be detected directly by nucleic acid analysis and sequences according to the invention (genomic DNA, RNA, or cDNA), but also via the polypeptides according to the invention.
  • nucleic acid analysis and sequences according to the invention genomic DNA, RNA, or cDNA
  • polypeptides according to the invention the use of an antibody according to the invention which recognizes an epitope carrying a mutation makes it possible to discriminate between a "healthy" protein and a protein "Associated with a pathology".
  • IBD1 is potentially important in many inflammatory diseases and / or immune. This result is to be compared with the fact that the pericentromeric region of chromosome 16 has been described as containing susceptibility genes to various human diseases such as ankylosing spondylitis or psoriatic arthritis. It can therefore be considered that IBD1 has an important role in a large number of inflammatory and / or immune diseases.
  • IBD1 can be associated with inflammatory diseases granulomatous.
  • Blau Syndrome and CD are diseases part of this family. So we hope to find variations in the IBD1 gene for other diseases of the same family (sarcoidosis, Behçet's disease ).
  • IBD1 in part explain this susceptibility to cancer and define new pathways of carcinogenesis.
  • the precise description of the mutations observable in the IBD1 gene allows thus laying the foundation for a molecular diagnosis of inflammatory diseases and where his role is demonstrated. Such an approach, based on the search for mutations in the gene, will contribute to the diagnosis of these diseases and possibly to reduce the importance of certain complementary examinations invasive or expensive.
  • the invention lays the foundation for such a molecular diagnosis based on on the search for mutations in IBD1.
  • Molecular diagnosis of inflammatory diseases should also be to improve the nosological classification of these diseases and to improve the define subgroups of particular patients by their clinical characteristics, the evolution of the disease or the response to certain treatments. For example, the dismemberment of existing mutations could thus make it possible to classify currently undetermined colitis that accounts for more than 10% of all inflammatory bowel.
  • Such an approach will make it possible to propose a early charge adapted to each patient. In general, such a approach makes it possible to hope to eventually define a individualized disease, according to the genetic terrain of each patient, including curative and preventive measures.
  • a method of diagnosis and / or evaluation is preferred.
  • prognosis of an inflammatory disease or a cancer characterized in that determines from a biological sample of a patient the presence of at least mutation and / or alteration of expression of the gene corresponding to SEQ ID No. 1 by analyzing all or part of a nucleic sequence corresponding to said gene.
  • the gene SEQ ID No. 3 can also be studied.
  • This method of diagnosis and / or prognostic evaluation can be used preventively (study of a predisposition to these inflammatory diseases or to cancer), or for the purpose of establishing and / or confirming a condition clinic in a patient.
  • the inflammatory disease is an inflammatory disease of digestive tract
  • cancer is a cancer of the digestive tract (small intestine or colon).
  • the teaching of the invention makes it possible to know the mutations presenting a linkage disequilibrium with inflammatory diseases of the tube digestive and therefore associated with such diseases.
  • the analysis may be performed by sequence of all or part of the gene, or by other methods known to those skilled in the art.
  • the analysis can also be performed by fixing a probe according to the invention corresponding to one of the sequences SEQ ID No. 1 or 3, on a DNA chip and hybridization on these microplates.
  • a DNA chip containing a sequence according to the invention is also one of the objects of the invention.
  • a protein chip containing an amino acid sequence according to the invention is also an object of the invention.
  • a protein chip allows the study of the interactions between the polypeptides according to the invention and other proteins or chemical compounds, and thus may be useful for the screening of compounds interacting with the polypeptides according to the invention.
  • the skilled person also knows how to implement techniques. allowing the study of the alteration of the expression of a gene, for example by the study mRNA (in particular by Northem Blot or by RT-PCR experiments, with probes or primers according to the invention), or expressed protein, in particularly by Western Blot, using antibodies according to the invention.
  • mRNA in particular by Northem Blot or by RT-PCR experiments, with probes or primers according to the invention
  • expressed protein in particularly by Western Blot, using antibodies according to the invention.
  • the gene tested is preferably the gene of sequence SEQ ID No. 1, the disease inflammatory drug for which we are trying to predict susceptibility being a disease of the gastrointestinal tract, particularly Crohn's disease, or ulcerative colitis. If one is trying to detect cancer, it is preferably colon cancer.
  • Such a method may be followed by a sequence step of all or part of the nucleic acid coding for the IBD1 protein, which makes it possible to predict the susceptibility to inflammatory disease or cancer.
  • the agent capable of specifically detecting a nucleic acid coding for the IBD1 protein is advantageously an oligonucleotide probe according to the invention, which may be formed of DNA, RNA, PNA, modified or not.
  • the modifications may include radioactive or fluorescent labeling, or be due to changes in the bonds between the bases (phosphorothioates, or methylphosphonates for example).
  • the person skilled in the art knows the protocols to isolate a specific DNA sequence.
  • Step b) of the above process described can also be an amplification step as described previously.
  • the invention also relates to a method for detecting and / or dosing of a nucleic acid according to the invention in a biological sample, comprising the following steps of contacting a probe according to the invention with a biological sample and detection and / or assay of the hybrid formed between said polynucleotide and the nucleic acid of the biological sample.
  • Such a kit may also contain positive or negative controls to ensure the quality of the results obtained.
  • the skilled person can also perform an amplification step using primers selected from the sequences according to the invention.
  • the invention also relates to compounds selected from an acid nucleic acid, a polypeptide, a vector, a cell, or an antibody according to the invention, or the compounds obtained by the screening methods according to the invention, as a medicament, particularly for the prevention and / or treatment of a inflammatory and / or immune disease or cancer, associated with the presence of less a mutation of the gene corresponding to SEQ ID No. 1 of preferably an inflammatory disease of the digestive tract, in particular Crohn's or ulcerative colitis.
  • the first step towards identifying the IBD1 gene was to reduce the size of the genetic region of interest, initially centered on the D16S411 marker located between D16S409 and D16S419 (Hugot et al., 1996 and Fig. 1).
  • a group of markers relatives was used to further clarify the genetic region and has made it possible to complete the genetic linkage and to look for an imbalance of genetic link with the disease.
  • SEQ ID NO: 50 D16S419 20.4 SEQ ID NO: 51 (AFM225zf2) SEQ ID NO: 52 D16S771 21.8 SEQ ID NO: 53 (GGAA23C09) SEQ ID NO: 54 D16S408 25.6 SEQ ID NO: 55 (AFM137xf8) SEQ ID NO: 56 D16S508 38.4 SEQ ID NO: 57 (AFPM304xf1) SEQ ID NO: 58
  • Each marker is listed according to the international nomenclature and the more often by the name proposed by the laboratory of origin. Markers appear in their order on the chromosome (from 16p to 16q). The distance between the markers (in centiMorgan Kosambi, calculated by the Crimap program from the experimental data) is indicated in the second column. The first polymorphic marker is arbitrarily taken as benchmark. Oligonucleotides used for the polymerization reaction in chain (PCR) are indicated in the third column.
  • the genotyping of these microsatellite markers was based on the technology of automatic sequencers using fluorescent primers. Briefly, after amplification, the fluorescent chain polymerization reaction (PCR) products were deposited on a polyacrylamide gel on an automatic sequencer according to the manufacturer's recommendations (Perkin Elmer). The size of the alleles for each subject was deduced thanks to Genescan R and Genotyper R software. The data was then stored on an integrated computer database containing genealogical, phenotypic and genetic data. They were then used for genetic linkage analyzes.
  • PCR fluorescent chain polymerization reaction
  • BAC artificial chromosomes of bacteria
  • a BAC contig containing 101 BACs was constructed and extending over an overall distance of more than 2.5 Mb with an average redundancy of 5.5 BAC at each point of the contig.
  • the average size of the BAC is 136kb.
  • BAC DNA was fragmented by sonication.
  • the DNA fragments thus generated were subjected to agarose gel electrophoresis and those larger than 1.5 kb were elected for analysis. These fragments were then cloned into phage m13 itself introduced into bacteria made competent by electroporation. After culture, clones DNA was recovered and sequenced by automatic sequencing methods using of fluorescent primers of the vector m13 on automatic sequencer.
  • SNPs biallelic polymorphism markers
  • the typing data was then analyzed according to an imbalance test of transmission (computer program TDT of the software GENEHUNTER version 2).
  • TDT computer program
  • GENEHUNTER version 2 For families with multiple affected relatives, only one patient has been taken into account for the analysis. Indeed, taking into account several patients related issues raises the problem of non-independence of data in calculations statistics and can induce inflation of the value of the test.
  • the patient serving the analysis was randomly drawn within each family by an automatic procedure of randomization. Given this randomization, the value of the statistical test obtained represented only one possible sample from the family group studied. In order not to limit the analysis to only this possible sample and to better understand the robustness of the results obtained, for each test, a Hundreds of random samples were generated and analyzed.
  • the markers were studied separately and then grouped according to their order on the chromosomal segment (KIAA0849ex9 (locus 1), hb27G11F (locus 2), Ctg22Ex1 (locus 3), SNP1 (locus 4), ctg2931-3ac / ola (locus 5), ctg2931-5ag / ola (locus 6), SNP3-2931 (locus 7), Ctg25Ex1 (locus 8), CTG35ExA (locus 9), ctg35ExC (locus 10), d16s3136 (locus 11), hb133D1f (locus 12), D16S3035 (locus 13), ADCY7int7 (locus 14)) (Table 2). Haplotypes with 2, 3 and 4 consecutive markers were analyzed using the same strategy (100 random samples taking for each family only one affected individual).
  • the highest risk susceptibility haplotype is defined by loci 7 to 10. It this is the 1-2-1-2 haplotype (Table 2).
  • the markers tested are, as expected, most often out of balance link between them.
  • the EST groups (Unigene references: Hs 135201, Hs87280, Hs122983, Hs146128, Hs105242, Hs116424, Hs61309, Hs151708, Hs 87296 and Hs132289) published and present on the BAC hb87b10 were studied in research a more complete complementary DNA sequence (cDNA).
  • cDNA complementary DNA sequence
  • IBD1prox clones available in public banks were sequenced and the sequences organized between them.
  • IBD1 a DNA library complementary to blood peripheral (Stratagene human blood cDNA lambda zapexpress ref 938202) was screened by PCR products generated from known TSEs according to the modalities proposed by the manufacturer. The sequence of the cDNAs thus identified was then used to a new screening of the cDNA library and so on until we obtain the presented cDNA.
  • EST hs135201 identified a non-listed cDNA available genetic databases (Genbank) It therefore corresponds to a new human gene. Comparison of cDNA sequence and DNA Genomics has shown that this gene consists of 11 exons and 10 introns. An exon additional, in 5 'position relative to the identified cDNA is predicted by the analysis of the sequence with the Grail software. These exons are very homologous with the first exons of the CARD4 / NOD1 gene. Considering all the exons identified and the additional putative exon, this new gene appears to have a genomic structure very close to that of CARD4 / NOD1. In addition, upstream the first putative exon is a transcription initiation site.
  • the putative exon was considered to be participating in this new gene.
  • the cDNA reported in the appendix (SEQ ID No. 1) thus comprises all of the sequence identified plus the sequence predicted by computer modeling, complementary DNA starting arbitrarily at the first ATG codon of the coding sequence predicted. On this basis, the gene would therefore have 12 exons and 11 introns.
  • the intron-exon structure of the gene is reported in SEQ ID NO: 3.
  • the protein sequence deduced from the nucleotide sequence comprises 1041 amino acids (SEQ ID NO: 2). This sequence was also not found on the biological databases (Genpept, pir, swissprot).
  • the IBD1 gene therefore has only 11 exons and 10 introns and code for a protein of 1013 amino acids (ie 28 less amino acids) initially determined).
  • the LRR domain of the protein makes it possible to affiliate the protein with a family of proteins involved in intracellular signaling and present both in plants and animals.
  • CARD4 / NOD1 The similarity between this new gene and CARD4 / NOD1 suggests that, as CARD4 / NOD1, the IBD1 protein is involved in the regulation of apoptosis and activation of NF-kappa B (Bertin et al., 1999, Inohara et al., 1999).
  • the regulation of cell apoptosis and activation of NF-kappa B are pathways of Intracellular signaling essential in immune reactions. Indeed, these signal transduction pathways are the effector pathways of the proteins of the TNF receptor family (Tumor Necrosis Factor) involved in cell-cell interactions and the cellular response to different mediators of inflammation (cytokines).
  • TNF receptor family Tumor Necrosis Factor
  • cytokines mediators of inflammation
  • NF-kB plays a pivotal role in diseases inflammatory bowel disease and is activated in the mononuclear cells of the lamina intestinal in Crohn's disease (Schreiber et al., 1998).
  • the Treatment of Crohn's disease is based on the use of sulfasalazine and glucocorticoids, both known to be NF-kB inhibitors (Auphan et al., 1995 and Wahl et al., 1998)
  • the candidate gene IBD1 code for a protein very similar to NOD2, a member of the superfamily CED4 / APAF1 (Ogura et al., 2000).
  • the nucleotide and protein sequences of IBD1 and NOD2 actually diverge only for a very small initial portion of 2 sequences reported.
  • the tissue expressions of Nod2 and IBD1 are moreover superimposed. These two genes (proteins) can therefore be considered as identical.
  • the Nod2 LRR domain has a binding activity for bacterial lipopolysaccharides (LPS) (Inohara et al., 2000) and that its Deletion stimulates the NFkB pathway. This result confirms the data of the invention.
  • LPS bacterial lipopolysaccharides
  • the tissue expression of IBD1 was then studied by the technique of Northern blot.
  • a 4.5 kb transcript is visible in most human tissues.
  • the size of the transcript is consistent with the size predicted by the cDNA.
  • the transcript of 4.5 kb appears in very low abundance in the small intestine and colon. It is by against very strongly expressed in white blood cells. This is in agreement with clinical data on transplants that suggest that Crohn's disease is potentially a disease related to circulating immune cells. Indeed, the intestinal transplantation does not prevent recurrence on the graft in the disease of Crohn's while bone marrow transplantation appears to be having an effect beneficial on the evolution of the disease.
  • the promoter of the IBD1 gene is currently not precisely identified. However, it is reasonable to think, by analogy with a very large number of genes that it resides, at least in part, immediately upstream of the gene, in the 5 'portion of it.
  • This genetic region contains sequences transcribed as evidenced by the presence of EST (HUMGS01037, AA835524, hs.105242, SHGC17274, hs.146128, hs.122983, hs.87280). ATCC clones containing these sequences were sequenced and analyzed in the laboratory, to highlight an organization in exons and introns with possible alternative splicing. These data suggest the existence of another gene (named IBD1prox because of its proximity to IBD1). The partial sequence of the complementary DNA of IBD1prox is reported (SEQ ID No. 4) as well as its intron-exon structure on SEQ ID NO: 6.
  • IBD1prox is the anonymous EST HUMGS01037 whose RNA is expressed more importantly in the differentiated leukocyte lineages than in the undifferentiated lineages.
  • this gene may have a role in inflammation and cell differentiation. It can therefore be considered as a good candidate for susceptibility to IBD.
  • the association between MC and the ctg35 ExC polymorphism localized on the coding sequence of IBD1prox enhances this hypothesis even if this polymorphism does not lead to sequence variation at the protein level.
  • IBD1 and IBD1prox are structurally related.
  • the strong proximity between the two genes may reflect a interaction between them.
  • the "head-tip" localization of these genes suggests that they may have common or interdependent modes of regulation.
  • the coding sequence and the intron-exon junctions of the gene were sequenced from the exon 2 in exon 12 included in 70 independent subjects, namely: 50 patients with MC, 10 patients with UC, 1 patient with Blau syndrome and 9 healthy witnesses.
  • the patients studied were for the most part family forms of disease and often carried the susceptibility haplotype defined by the transmission imbalance studies. Healthy witnesses were original caucasian.
  • the mutations other than silent observed in each exon are reported. They are indicated by the variation of the peptide chain. For each mutation and for each phenotype studied, the number of times where the mutation is observed, relative to the number of chromosomes tested.
  • non-silent variants were present both in patients with RCH or MC and in healthy subjects. These were S269P variants, N290S, R703W and V956I located in exons 2, 4 and 9. A complement information seems necessary before retaining a possible functional role to these sequence variants.
  • V956I is a conservative sequence variation (amino acids aliphatic).
  • the S269P sequence variant corresponds to a variation of acid class amine (hydroxylated immunoacid) at the beginning of the nucleotide binding domain. It imbalance of transmission with MC. It is indeed the polymorphism Snp3 (See above).
  • R703W results in a modification of the class of amino acid (aromatic instead of basic). This change occurs in the intermediate region between the NBD and LRR domains, a region conserved between IBD1 and CARD4 / NOD1. A role functional can be suspected for this polymorphism. This variation of sequence (corresponding to the Snp1 polymorphic site) is more often transmitted to the patients with CD that chance does not want (see above) confirming that this polymorphism is associated with MC. It is possible that the presence of this mutant in healthy subjects demonstrates incomplete penetrance of the mutation as this is expected for complex genetic diseases such as diseases chronic inflammatory bowel.
  • the variant R704C located immediately next to R703W could be identified both in CM and in UC. It also corresponds to a variation not conservative protein (sulfur amino acid instead of basic) on the same protein region, suggesting a functional effect as important for R704C as for R703W.
  • the G909R mutation occurs on the last amino acid of the sixth motif PBA. It replaces an aliphatic amino acid to a basic amino acid. This variation is potentially significant given the character usually neutral or polar terminal amino acids rich leucine motifs (for both IBD1 and NOD1 / CARD4) and the preserved nature of this acid amino acid on IBD1 and NOD1 / CARD4 proteins.
  • the gene promoter is currently not determined. According to any likelihood, however, this is probably located in the 5 'upstream region of the gene. According to this hypothesis, the sequence variants observed in this region can have a functional effect. This could explain the very strong association between MC and some polymorphic loci such as ctg35 ExC or Ctg25Ex1.
  • the invention thus provides the first description of mutations in the gene family containing a CARD domain in humans.
  • the frequency of these mutations in various inflammatory diseases shows that the IBD1 gene has a essential role in the normal and pathological inflammatory process.
  • This invention provides new ways of understanding and research in the field of physiopathology of normal inflammatory processes and pathological. It allows to consider the development of new pharmaceutical molecules regulating the effector pathways controlled by IBD1 and useful in the treatment of inflammatory diseases and the regulation of the process inflammatory in general.
  • V928I (formerly V956I) is not significantly associated with any of the Inflammatory bowel diseases and therefore does not seem to have a significant role in the disease.
  • S241P mutation (formerly S269P) is in link disequilibrium with other major changes and does not seem to play a role in itself important in susceptibility to inflammatory bowel disease (data not shown).
  • the IBD1prox gene located in the promoter region of IBD1, and whose partial sequence is disclosed in the present invention, can also have a important role in regulating cellular apoptosis and the process inflammatory, as suggested by its differential expression in cells mature immune system.
  • Inflammatory bowel diseases are genetic diseases complexes for which, to date, no susceptibility gene had been identified with certainty.
  • the invention has made it possible to identify the first gene of Susceptibility to Crohn's disease by a positional cloning approach (or reverse genetics). This is the first genetic location obtained by a approach for a complex genetic disease, demonstrating its usefulness and its feasibility, at least in some cases in complex genetic diseases.
  • the present invention also relates to a purified or isolated nucleic acid characterized in that it encodes a polypeptide having a continuous fragment of at least at least 200 amino acids of a protein selected from SEQ ID No. 2 and SEQ ID No. 5.

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Claims (22)

  1. Gereinigte oder isolierte Nukleinsäure, dadurch gekennzeichnet, dass sie eine Nukleinsäuresequenz umfasst, welche ausgewählt wird aus der Gruppe von folgenden Sequenzen:
    a) SEQ ID NO.1 und SEQ ID NO.3;
    b) der komplementären Sequenz oder der RNA-Sequenz, welche einer Sequenz, wie in a) definiert, entspricht.
  2. Gereinigte oder isolierte Nukleinsäure nach Anspruch 1, dadurch gekennzeichnet, dass sie folgendes umfasst oder aus folgendem besteht: SEQ ID NO. 1, die komplementäre Sequenz, oder die dieser Sequenz entsprechende RNA-Sequenz.
  3. Isoliertes Polypeptid, dadurch gekennzeichnet, dass es ein Polypeptid, welches SEQ ID NO.2 entspricht, umfasst.
  4. Polypeptid nach Anspruch 3, dadurch gekennzeichnet, dass es aus SEQ ID NO.2 besteht.
  5. Klonierungs- und/oder Expressionsvektor, welcher eine Nukleinsäure nach einem der Ansprüche 1 und 2 oder eine, die ein Polypeptid nach einem der Ansprüche 3 und 4 kodiert, umfasst.
  6. Wirtszelle, dadurch gekennzeichnet, dass sie durch einen Vektor nach Anspruch 5 transformiert ist.
  7. Tier, mit Ausnahme des Menschen, dadurch gekennzeichnet, dass es eine Zelle nach Anspruch 6 umfasst.
  8. In vitro-Verwendung einer Nukleinsäure nach einem der Ansprüche 1 und 2 als Sinn- oder Antisinn-Nukleotid.
  9. Verwendung einer Nukleinsäuresequenz nach einem der Ansprüche 1 und 2 für die Herstellung eines rekombinanten Polypeptids.
  10. Verfahren zur Herstellung eines rekombinanten Polypeptids, dadurch gekennzeichnet, dass man eine Zelle nach Anspruch 6 unter Bedingungen, welche die Expression des Polypeptids erlauben, kultiviert und dass man das rekombinante Polypeptid gewinnt.
  11. Monoklonaler oder polyklonaler Antikörper, dadurch gekennzeichnet, dass er selektiv ein Polypeptid nach Anspruch 4 bindet.
  12. Verfahren zum Nachweis eines Polypeptids nach einem der Ansprüche 3 und 4, dadurch gekennzeichnet, dass es die folgenden Schritte umfasst:
    a) Inkontaktbringen einer biologischen Probe mit einem Antikörper nach Anspruch 11;
    b) Nachweisen des gebildeten Antigen-Antikörper-Komplexes.
  13. Reagenzienkit für das Ausführen eines Verfahrens nach Anspruch 12, dadurch gekennzeichnet, dass er umfasst:
    a) einen monoklonalen oder polyklonalen Antikörper nach Anspruch 11;
    b) gegebenenfalls Reagenzien für die Bildung eines Mediums, welches für die immunologische Reaktion günstig ist;
    c) die Reagenzien, die den Nachweis des während der immunologischen Reaktion erzeugten Antigen-Antikörper-Komplexes erlauben.
  14. Diagnose- und/oder prognostisches Bewertungsverfahren einer entzündlichen und/oder das Immunsystem betreffenden Erkrankung oder einer Krebserkrankung, dadurch gekennzeichnet, dass man ausgehend von einer biologischen Probe eines Patienten das Vorhandensein von wenigstens einer Mutation und/oder eine Veränderung der Expression des SEQ ID NO.1 oder SEQ ID NO.3 entsprechenden Gens durch die Analyse der Gesamtheit oder eines Teils einer Nukleinsäuresequenz, welche dem Gen entspricht, bestimmt.
  15. DNA-Chip, dadurch gekennzeichnet, dass er eine Nukleinsäuresequenz nach einem der Ansprüche 1 und 2 enthält.
  16. Chip mit Proteinen, dadurch gekennzeichnet, dass er ein Polypeptid nach einem der Ansprüche 3 und 4 oder einen Antikörper nach Anspruch 11 enthält.
  17. Verfahren zum Nachweis und/oder zur quantitativen Bestimmung einer Nukleinsäure nach einem der Ansprüche 1 und 2 in einer biologischen Probe, dadurch gekennzeichnet, dass es die folgenden Schritte umfasst:
    a) Inkontaktbringen eines markierten Polynukleotids nach einem der Ansprüche 1 und 2;
    b) Nachweis und/oder quantitative Bestimmung des zwischen dem Polynukleotid und der Nukleinsäure der biologischen Probe gebildeten Hybrids.
  18. Verfahren zum Nachweis und/oder zur quantitativen Bestimmung einer Nukleinsäure nach einem der Ansprüche 1 und 2 in einer biologischen Probe, dadurch gekennzeichnet, dass es einen Amplifizierungsschritt der Nukleinsäuren der biologischen Probe mit Hilfe von Primern, die Fragmenten von wenigstens 15 aufeinanderfolgenden Nukleotiden von SEQ ID NO.1 oder SEQ ID NO.3 entsprechen, umfasst.
  19. Verfahren zum Screenen auf Verbindungen, die in der Lage sind, an ein Polypeptid mit der Sequenz SEQ ID NO.2 zu binden, dadurch gekennzeichnet, dass es die Schritte umfasst, ein Polypeptid nach einem der Ansprüche 3 und 4, eine Zelle nach Anspruch 6 oder ein Säugetier nach Anspruch 7 mit einer Kandidatenverbindung in Kontakt zu bringen und die Bildung eines Komplexes zwischen der Kandidatenverbindung und dem Polypeptid nachzuweisen.
  20. Verfahren zum Screenen auf Verbindungen, die zur Wechselwirkung in vitro oder in vivo mit einer Nukleinsäure nach einem der Ansprüche 1 und 2 in der Lage sind, dadurch gekennzeichnet, dass es die Schritte umfasst, eine Nukleinsäure nach einem der Ansprüche 1 und 2, eine Zelle nach Anspruch 6 oder ein Säugetier nach Anspruch 7 mit einer Kandidatenverbindung in Kontakt zu bringen und die Bildung eines Komplexes zwischen der Kandidatenverbindung und der Nukleinsäure nachzuweisen.
  21. Verbindung, dadurch gekennzeichnet, dass sie ausgewählt wird aus
    a) einer Nukleinsäure nach einem der Ansprüche 1 und 2;
    b) einem Polypeptid nach einem der Ansprüche 3 und 4;
    c) einem Vektor nach Anspruch 5;
    d) einer Zelle nach Anspruch 6; und
    e) einem Antikörper nach Anspruch 11;
    als Arzneimittel.
  22. Verbindung nach Anspruch 21 für die Verhütung und/oder die Behandlung einer entzündlichen und/oder das Immunsystem betreffenden Erkrankung oder einer Krebserkrankung, welche mit dem Vorhandensein von wenigstens einer Mutation des SEQ ID NO.1 entsprechenden Gens verbunden ist.
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CA2404448A1 (fr) 2001-10-04
US8137915B2 (en) 2012-03-20
US20030190639A1 (en) 2003-10-09
WO2001072822A3 (fr) 2002-07-18
WO2001072822B1 (fr) 2002-09-06
WO2001072822A2 (fr) 2001-10-04
US7592437B2 (en) 2009-09-22
AU2001287272B2 (en) 2006-09-21
CA2404448C (fr) 2014-03-25
FR2806739A1 (fr) 2001-09-28
US20100159453A1 (en) 2010-06-24
DE60109430T2 (de) 2006-04-06
ATE291086T1 (de) 2005-04-15
DE60109430D1 (de) 2005-04-21
EP1268784A2 (de) 2003-01-02
JP2003528631A (ja) 2003-09-30
ZA200207821B (en) 2003-06-12
US20130035260A1 (en) 2013-02-07
NZ522011A (en) 2005-11-25
FR2806739B1 (fr) 2005-02-18
AU8727201A (en) 2001-10-08
JP5150997B2 (ja) 2013-02-27

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